Gastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocyte generation is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae (Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immune challenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata. Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in the blood of conspecific recipients. We tracked cell proliferation by labeling the donors' hemocytes with the fluorescent cell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeled hemocytes survived and proliferated into the recipients’ circulation for at least 17 days. We also determined the cell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) staining methods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), while a lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, we further distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. We regarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings by assessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First, we used JC-1 staining to determine the mitochondrial membrane potential (Ψ_m) of circulating hemocytes, which is expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicates a high Ψ_m. Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thus indicating a low Ψ_m. It is known that the transition from a quiescent to a proliferating state associates with an increase of the Ψ_m. The specificity of these changes was here controlled by membrane depolarization with the Ψ_m disruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABC transporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells. Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06–0.12%) showed a high dye-efflux activity, which was significantly inhibited by 100 and 500 μM verapamil, and thus is indicative of an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, among circulating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave as progenitor cells later, either in the circulation or the hematopoietic tissues/organs.

腹足类动物的造血作用发生在某些物种的特殊组织中，但证据还表明，血红细胞的生成可能在结缔组织或血液系统的其他物种中广泛分布。在壶腹科（Caenogastropoda）中，肾脏和肺都含有假定的造血细胞，它们会对免疫挑战作出反应。在当前的研究中，我们想探讨在Pomacea canaliculata的血液中是否发生造血作用。因此，我们从供体动物获得了循环血细胞，并测试了它们在同种受体的血液中增殖的能力。我们通过用荧光细胞增殖标记物羧荧光素二乙酸琥珀酰亚胺酯（CFSE）标记供体的血细胞来跟踪细胞增殖。转移的CFSE标记的血细胞存活并增殖进入受体循环至少17天。我们还通过使用碘化丙啶（PI）和a啶橙（AO）染色方法确定了循环血细胞的细胞周期状态。流式细胞仪分析显示，大多数PI染色的血细胞处于G1期（〜96％），而较少的细胞通过G2 / SM过渡（〜4％）。当我们改为使用AO染色时，我们进一步区分出了小细胞（约5％）的亚群，复杂性，粒度和RNA含量。我们将此亚群视为静态细胞。在单独的实验组中，我们通过在循环血细胞中评估静态，未分化细胞的两个进化保守特征来补充这些发现。首先，我们使用JC-1染色确定线粒体膜电位（Ψ_m）循环血细胞，预计在静止细胞中较低。大多数血细胞（〜87％）显示出JC-1，这表明高的Ψ高聚合_米。除此之外，少量的血细胞亚群（〜11％）显示出较低的染料聚集，因此表明indicating _m低。已知的是，从静止到增殖状态相关联的过渡随着的Ψ_米。这些变化的特异性这里由细胞膜去极化与Ψ控制_米破坏者CCCP。其次，我们用Hoechst33342染料对血细胞进行染色，以确定ABC转运蛋白的外排活性，ABC转运蛋白参与未分化细胞的多异生物抗性系统。大多数血细胞（> 99％）显示出较低的染料流出活性，但一小部分细胞（0.06-0.12％）显示出较高的染料流出活性，这被100和500μM维拉帕米显着抑制，因此是指示性的循环血细胞的未分化亚群。综上所述，我们的结果表明，在循环血细胞中，有一些细胞能够在循环系统或造血组织/器官中增殖或保持静止状态，并在以后充当祖细胞。