Università degli studi di Modena e Reggio Emilia

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.

Histological studies focusing on the early cephalic tentacle regeneration in P. canaliculata, have demonstrated that wound closure and blastema formation took place within 24 h post amputation (hpa). A Matlab® plugin allowed the semi-automated identification and quantification of a phagocytic hemocyte sub-population in the blastema. Flow cytometry analysis showed that the injection of the phagocyte-specific drug Clophosome® (45 µg/g snail) could transiently remove circulating hemocytes, that recovered the pre-treatment level within 24 h. Consistently, histological experiment demonstrated that rare hemocytes were present in the early regenerating tentacles of Clophosome®-injected snails

In humans, injuries and diseases can result in irreversible tissue or organ loss. This wellknown 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.

Pomacea canaliculata is a freshwater gastropod known for being both a highly invasive species and one of the possible intermediate hosts of the mammalian parasite Angiostrongylus cantonensis. With the aim of providing new information concerning P. canaliculata biology and adaptability, the first proteome of the ampulla, i.e., a small organ associated with the circulatory system and known as a reservoir of nitrogen-containing compounds, was obtained. The ampullar proteome was derived from ampullae of control snails or after exposure to a nematode-based molluscicide, known for killing snails in a dose-and temperature-dependent fashion. Proteome analysis revealed that the composition of connective ampulla walls, cell metabolism and oxidative stress response were affected by the biopesticide. Ultrastructural investigations have highlighted the presence of rhogocytes within the ampullar walls, as it has been reported for other organs containing nitrogen storage tissue. Collected data suggested that the ampulla may belong to a network of organs involved in controlling and facing oxidative stress in different situations. The response against the nematode-based molluscicide recalled the response set up during early arousal after aestivation and hibernation, thus encouraging the hypothesis that metabolic pathways and antioxidant defences promoting amphibiousness could also prove useful in facing other challenges stimulating an oxidative stress response, e.g., immune challenges or biocide exposure. Targeting the oxidative stress resistance of P. canaliculata may prove helpful for increasing its susceptibility to bio-pesticides and may help the sustainable control of this pest’s diffusion.

Simple SummarySustainable solutions to the spreading of invasive species are difficult to find due to the absence of biological information about basic immune mechanisms of the target pests. Here, we present evidence of the effects of a commercially available roundworm, Phasmarhabditis hermaphrodita, against the invasive apple snail Pomacea canaliculata. The effects are principally evaluated in terms of snail survival and immune activation. Via molecular and microscopy-based approaches, we demonstrate that dosage and temperature are critical in determining the effects of the roundworm, and that the apple snail response to this immune challenge involves different organs. To our knowledge, these findings are the first demonstration that a P. hermaphrodita-based molluscicide can effectively kill P. canaliculata and that the snail can mount a multi-organ response against this pathogenic roundworm.The spreading of alien and invasive species poses new challenges for the ecosystem services, the sustainable production of food, and human well-being. Unveiling and targeting the immune system of invasive species can prove helpful for basic and applied research. Here, we present evidence that a nematode (Phasmarhabditis hermaphrodita)-based molluscicide exerts dose-dependent lethal effects on the golden apple snail, Pomacea canaliculata. When used at 1.7 g/L, this biopesticide kills about 30% of snails within one week and promotes a change in the expression of Pc-bpi, an orthologue of mammalian bactericidal/permeability increasing protein (BPI). Changes in Pc-bpi expression, as monitored by quantitative PCR (qPCR), occurred in two immune-related organs, namely the anterior kidney and the gills, after exposure at 18 and 25 degrees C, respectively. Histological analyses revealed the presence of the nematode in the snail anterior kidney and the gills at both 18 and 25 degrees C. The mantle and the central nervous system had a stable Pc-bpi expression and seemed not affected by the nematodes. Fluorescence in situ hybridization (FISH) experiments demonstrated the expression of Pc-bpi in circulating hemocytes, nurturing the possibility that increased Pc-bpi expression in the anterior kidney and gills may be due to the hemocytes patrolling the organs. While suggesting that P. hermaphrodita-based biopesticides enable the sustainable control of P. canaliculata spread, our experiments also unveiled an organ-specific and temperature-dependent response in the snails exposed to the nematodes. Overall, our data indicate that, after exposure to a pathogen, the snail P. canaliculata can mount a complex, multi-organ innate immune response.