The Origin of Hyperspread Cells, a Group of Circulating Hemocytes in Mythimna separata

In Mythimna separata (Ms) larvae, a hyperspread cell (HSC) has been identified as a new type of hemocyte, in addition to the existing five types of hemocytes: prohemocytes, granulocytes, plasmatocytes, spherulocytes, and oenocytoids. HSCs adhere to invading foreign substances, deposit melanin granules, and promote the adhesion of other hemocytes, thereby playing an important role in the early phase of cellular immune response, such as encapsulation and nodulation. However, the origin of HSCs remained unclear. Nodulation caused by artificial injection of a large number of yeast cells lowered HSC population density temporarily but later recovered to keep 2%-5% among total hemocytes, suggesting continuous de novo supply from specific source tissues. The origin of HSCs was examined by some possibilities as follows: (1) self-renewal, (2) the existing circulating hemocytes, and (3) hematopoietic organs. Propidium iodide staining confirmed that HSCs undergo cell death following immune reactions on foreign surfaces, ruling out self-renewal possibility as the origin. Further, circulating hemocytes were fractionated based on adhesive specificity. In vitro incubation revealed that the non-adherent fraction (prohemocytes, spherulocytes, and oenocytoids) produced HSCs, whereas the adherent fraction (granulocytes and plasmatocytes) did not. Separation using a discontinuous Percoll cushion demonstrated that the prohemocytes differentiated into HSCs. In vitro hematopoietic organs incubation released prohemocytes and other hemocytes gradually within 1-24 h; however, HSC differentiation occurred with a time lag. Moreover, hematopoietic organs extirpation did not affect the HSC population recovery after yeast-injection. These results showed that HSCs originate from prohemocytes released from hematopoietic organs in the circulation.