Figures: In Vitro Hemocyte Nodulation in a Molluscan Invertebrate: Morphological and Functional Insights

Abstract

The apple snail Pomacea canaliculata has emerged as a promising model organism for invertebrate immuno-biology due to its robust cellular defenses and broad ecological adaptability. Among its immune responses, hemocyte nodulation—a process forming spheroidal aggregates that entrap and neutralize patho-gens—represents a key but poorly understood phenomenon. Nodulation is a phylogenetically widespread process of hemocyte aggregations serving the encasing of intruders thereby avoiding their dissemination or, somehow paradoxically, to provide them shelters for their replication. Here, we characterize the spontaneous aggregation and nodulation of P. canaliculata hemocytes under axenic culture conditions using scanning electron microscopy (SEM), thereby revealing intrinsic properties of hemocyte behavior in the absence of microbial stimuli. Within minutes of culture initiation, hemocytes formed small aggregates (3–6 cells) inter-connected by fine cytoplasmic extensions that resembled tunneling nanotubes (TNT), which were characterized by nanometer-scale bridges suspended above the substrate, linking individual hemocytes or aggregates. These structures likely support intercellular communication and coordination during aggregation. Over subsequent hours, these aggregates exhibited three distinct morphotypes—flat mats, bundles, and spheroidal nod-ules—culminating in the formation of large, compact structures within 96 h. These configurations indicate a dynamic and self-organized process of interhemocyte interaction. Other key observations in this paper include the fusion of cytoplasmic extensions (particularly filopodia) between adjacent hemocytes, the emission of pointed projections (here called spines) that may elongate into TNT-like structures, the recruitment and in-corporation of hemocytes into developing nodules, and hemocyte fusion within the nodules. Our observations suggest that hemocyte nodulation is not merely a reaction to infection but an endogenously emergent behavior. The structural organization of nodules, along with the presence of TNT-like connections, supports the concept of “supercellularity,” wherein coordinated hemocytes behave as multicellular immune units. Such features may represent evolutionarily conserved elements of metazoan immune architecture. Collectively, this work provides unprecedented fine morphological details of in vitro hemocyte nodulation, offering a baseline for mechanismic and molecular investigations of invertebrate cellular immunity and highlighting the plausible homology of nodular immune structures across metazoans.