Circulating hemocytes continue to proliferate throughout lifespan in Daphnia

Abstract Daphnia is a classic model organism for the study of circulation and hemocyte biology. Despite the ease of observation and separation of hemocytes, it is not known whether the circulating hemocytes continue to be produced throughout Daphnia lifespan, and if yes, whether they are generated in any specific hemopoietic tissue or represent a reproducing population as known in some arthropods. We detected de novo DNA synthesis by means of EdU staining in a significant portion of circulating hemocytes in adult D. magna. This portion did not significantly vary among phases of molting/ovary cycle and showed only a slight decrease with age. We did not detect any matching newly synthesized DNA anywhere in any other tissues of adult daphnids, eliminating the hypothesis of the existence of a population of hemopoietic stem cells. Rather, it appears that circulating hemocytes are a reproducing population with mitoses continuing throughout life. Hemocytes showed average ploidy of 4, with a considerable variation around that value indicating that either some endomitoses were also present or a significant portion of hemocytes were sampled in G2. However, while there was no difference in total DNA content measured by Hoechst fluorescence between hemocytes that showed and did not show recent DNA synthesis, among EdU-positive hemocytes there was a positive correlation between Hoechst and EdU fluorescence, indicating that many cells were sampled during S-phase. It is not known whether the total number of hemocytes increases with age or the observed proliferation compensates for losses of hemocytes, as well as whether the EdU-positive hemocytes represent a separate mitotically active stem subpopulation of circulating hemocytes.