Nicola Franchi

In vertebrates, circulating and tissue-resident immune cells contribute to host defense and tissue homeostasis. Their balance is maintained through hematopoiesis and anatomical reservoirs. In mollusks, this balancing is poorly investigated. Thus, the dynamics of circulating and tissue-resident hemocytes after a hemolymph withdrawal were analyzed in Pomacea canaliculata, a snail that allows repeated hemolymph withdrawals and possesses posterior kidney (PK) cell aggregates known as hemocyte islets. In the absence of specific hemocyte markers, flow cytometry data revealed dynamic changes in circulating hemocyte number and populations within 48 h following hemolymph withdrawal. In addition, computer-assisted image analysis of histological sections showed a significant transient reduction in PK hemocyte-islet area. Analyses of publicly available P. canaliculata transcriptomes revealed predominant and highly variable expression of two hematopoiesis-related genes, namely Transglutaminase (PcTGase) and Hematopoietically expressed homeobox protein (PcHhex), in circulating hemocytes and PK. Fluorescence in situ hybridization highlighted constitutive PcTGase and PcHhex expression within PK hemocyte islets. RT-qPCR experiments confirmed transcriptomic data on PcTGase and PcHhex constitutive expression, without significant modulation following hemolymph withdrawal. Collectively, these findings provide the first evidence of parallel recovery dynamics involving circulating hemocytes and PK hemocyte islets in P. canaliculata.

The increasing fragmentation of plastic debris into nanosized particles represents a threat to freshwater ecosystems, yet the biological effects of nanoplastics (NPs) on freshwater invertebrates remain poorly understood. This study investigated tissue distribution, cellular effects and immune responses following acute exposure to polyethylene terephthalate nanoplastics (PET-NPs) in the freshwater gastropod Pomacea canaliculata, a species of high ecological relevance and physiological resilience. Adult snails were injected with PET-NPs at 5 or 10 mg/L and sampled after 24 and 72 h. PET-NPs accumulation in the anterior and posterior kidneys was assessed by fluorescence imaging and tissue morphology was evaluated. Stress- and inflammation-related genes (Pc-Heat Shock Protein (HSP)70, Pc-HSP90 and Pc-Allograft inflammatory factor 1) expression was quantified by RT-qPCR. PET-NPs uptake and phagocytic activity were analyzed in circulating hemocytes in vivo and ex vivo. PET-NPs were accumulated in renal tissues, persisting up to 72 h without histopathological alterations. Gene expression analyses revealed non-linear and dose/time-dependent responses. Hemocytes of different morphologies internalized PET-NPs in a dose-dependent manner and showed intercellular particle transfer. Overall, acute PET-NP exposure determines rapid immune handling and tissue sequestration with limited short-term physiological impact, underscoring the potential involvement of immune processes in NPs fate and highlighting the need for chronic exposure studies.

Allograft inflammatory factor-1 (AIF1) is a conserved calcium-binding protein involved in inflammatory and neuro-immune responses and expressed in Pomacea canaliculata (Pc-AIF1) during cephalic tentacle regeneration. Here, we investigated the expression and distribution of Pc-AIF1 in control conditions and during cephalic tentacle regeneration. A transcriptomic analysis of 315 RNA-seq datasets revealed maximal Pc-AIF1 expression in circulating hemocytes and hemocyte-rich tissues. Pc-AIF1 was also highly expressed in neural ganglia. Fluorescence in situ hybridization (FISH) evidenced Pc-AIF1 in circulating hemocytes and in the phagocytic hemocyte aggregates in the posterior kidney. qPCR showed the constitutive expression of Pc-AIF1 in cerebral ganglia. FISH experiments showed Pc-AIF1-positive cells within the cephalic tentacle blastema at 24 h post-amputation (hpa). Even if the amputation left them untouched, both the ipsilateral and contralateral cerebral ganglia increased Pc-AIF1 expression until 48 hpa. Immunocytochemical experiments evidenced positive cells to RCA120 (a microglial marker in mammals) among circulating hemocytes, in the connective tissue surrounding the cerebral ganglia, and within the regenerating tentacles. These findings suggest that Pc-AIF1 is a neuro-immune marker constitutively expressed in hemocyte populations and neural tissues; it is associated with the immediate hemocyte response to wounding and the neuro-immune interplay during the regeneration of sensory organs.

The knowledge of comparative and developmental immunobiology has grown over the years and has been strengthened by the contributions of multi-omics research. High-performance microscopy, flow cytometry, scRNA sequencing, and the increased capacity to handle complex data introduced by machine learning have allowed the uncovering of aspects of great complexity and diversity in invertebrate immunocytes, i.e., immune-related circulating cells, which until a few years ago could only be described in terms of morphology and basic cellular functions, such as phagocytosis or enzymatic activity. Today, invertebrate immunocytes are recognized as sophisticated biological entities, involved in host defense, stress response, wound healing, organ regeneration, but also in numerous functional aspects of organismal life not directly related to host defense, such as embryonic development, metamorphosis, and tissue homeostasis. The multiple functions of immunocytes do not always fit the description of invertebrate organisms as simplified biological systems compared to those represented by vertebrates. However, precisely the increasing complexity revealed by immunocytes makes invertebrate organisms increasingly suitable models for addressing biologically significant and specific questions, while continuing to present the undeniable advantages associated with their ethical and economic sustainability.

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.

Reactive oxygen species (ROS) are volatile and short-lived molecules playing important roles in several physiological functions, including immunity and physiological adaptation to unsuitable environmental conditions. In an eco-immunological view, the energetic costs associated with an advantageous metabolic apparatus able to cope with wide changes in environmental parameters, e.g., temperature range, water salinity or drought, could be further balanced by the advantages that this apparatus may also represent in other situations, e.g., during the immune response. This review provides an overview of molluscs included in the IUCN list of the worst invasive species, highlighting how their relevant capacity to manage ROS production during physiologically challenging situations can also be advantageously employed during the immune response. Current evidence suggests that a relevant capacity to buffer ROS action and their damaging consequences is advantageous in the face of both environmental and immunological challenges, and this may represent a trait for potential invasiveness. This should be considered in order to obtain or update information when investigating the potential of the invasiveness of emerging alien species, and also in view of ongoing climate changes.

By mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript for a novel styelin-like antimicrobial peptide, which we named botryllin. The gene is constitutively transcribed by circulating cytotoxic morula cells (MCs) as a pre-propeptide that is then cleaved to mature peptide. The synthetic peptide, obtained from in silico translation of the transcript, shows robust killing activity of bacterial and unicellular yeast cells, causing breakages of both the plasma membrane and the cell wall. Specific monoclonal antibodies were raised against the epitopes of the putative amino acid sequence of the propeptide and the mature peptide; in both cases, they label the MC granular content. Upon MC degranulation induced by the presence of nonself, the antibodies recognise the extracellular nets with entrapped bacteria nearby MC remains. The obtained results suggest that the botryllin gene carries the information for the synthesis of an AMP involved in the protection of B. schlosseri from invading foreign cells.