Analysis of the hemocyte population dynamics in Pomacea canaliculata using flow cytometry, microscopy and molecular biology-based approaches

Invertebrate immune systems have been found to be unexpectedly complex, with hemocytes being identified as key cells in invertebrate immunity, as well as in homeostasis and development. In molluscs, there is limited knowledge regarding the tissue context in which these cells can proliferate and mature, and no cell-specific molecular markers have been identified to date. The laboratory bred snail Pomacea canaliculata offers the possibility to stimulate the hematopoietic system by multiple hemolymph samplings. Its hemocytes have been categorized into small and blast-like Group I (GI) and large Group II (GII). The number of circulating hemocytes and the GII/GI ratio are stable and individual-specific, suggesting the presence of control mechanisms over hemocyte populations and cell-specific functions. In this regard, circulating hemocyte populations were assessed by flow cytometry 1.5, 3, 6, 9, 18, 24 or 48h following a previous hemolymph collection. After observing that the number of circulating hemocytes and the GII/GI ratio could be restored to their original state, both histological and fluorescent nuclear staining were performed on the hemocytes to check for the presence of mitotic figures. Then, DNA content in circulating hemocytes and in cells from the pericardial fluid (i.e. a recognized hematopoietic tissue in other gastropods) was evaluated via flow cytometry to check for DNA replication. Finally, to investigate the potential of circulating hemocytes to replicate at a molecular level, two cell proliferation markers were evaluated: the PCNA (Proliferating Cell Nuclear Antigen) protein and the proliferation marker protein Ki-67. An in-silico analysis was performed for both molecules, and their expression was evaluated in circulating hemocytes via RT-PCR. Only PCNA, was evaluated also via Western blot (WB). During hemolymph repopulation, circulating hemocyte populations exhibited the lowest GII/GI ratio 18h post-collection and recovered at 24h, when the total hemocyte number was significantly higher. It was not until 48h after the first collection that the original number of circulating hemocytes and the GII/GI ratio were restored. Morphological analysis did not suggest the presence of active mitosis in circulation, but interestingly, flow cytometry analysis showed that hemocytes had varying amounts of DNA unrelated to cell cycle and that there was a positive correlation between DNA-content and cell size. In agreement with the data on GII/GI ratio, high-DNA-content circulating hemocytes significantly decreased after 18h and returned to baseline after 24h, while no significant changes were seen among pericardial fluid cells. The observed data on DNA content might be explained by the phenomenon of cell endocycling, a process that has been documented in gastropod neurons and other invertebrate cell types, but not in molluscan hemocytes. Consistent with this idea, preliminary RT-PCR and WB data showed the expression of PCNA, a key eukaryotic DNA replication and repair factor, in control circulating hemocytes. These data suggest that P. canaliculata controls both the number of circulating hemocytes and the proportion of hemocyte populations. P. canaliculata circulating hemocytes appear to undergo DNA amplification unrelated to cell cycle, especially in larger cells. While the biological significance of this observation remains unclear, it further supports the potential of hemolymph repopulation as a promising context to study hemocyte population dynamics, turnover, and their specialization in controlling homeostasis and immune response.