Two hemocyte lineages exist in silkworm larval hematopoietic organ

Abstract

Background: Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster.

Methodology/principal findings: To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids.

Conclusions/significance: From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.

Figure 1. Ex vivo differentiation from progenitor cells in HPO.

CecB-GFP HPOs were enzymatically dispersed and injected into the hemocoel of non-transgenic larvae. Five days later, collected hemocytes were analyzed (A, B) and GFP-expressing cells were sorted (C–L). A: two-dimensional plots with PI/GFP of whole collected hemocytes. B: two-dimensional plots with FS/SS of whole collected hemocytes. Green and red dots are of GFP negative and positive cells, respectively. Clusters of spherulocytes (SP), granulocytes (GR), plasmatocytes (PL), and oenocytoids (OE) are gated according to Nakahara et al. (2009). C, D: All sorted cells fluoresce bright green, except for oenocytoids (yellow arrowheads) that had collapsed immediately after sorting. Among the GFP-expressing cells, prohemocytes (E, F), plasmatocytes (G, H), and granulocytes (I, J) were also observed. The sorted GFP+ cells were stained with anti-granulocyte antibody (K, L). Bar  = 20 µm (C), 10 µm (E, G, I), 40 µm (K).

Figure 2. In vitro culture of B. mori hemocyte-progenitor cells.

A: dispersed HPO cells viewed under a differential interference microscope. B: these cells were seeded into 24-well multiplates at a density of 2×105 cells/500 µl medium/well. C–H: the cells were cultured in medium containing 3% larval plasma (C, E, G) or plasma-free medium (D, F, H). Cells were viewed at 24 h (C, D), 72 h (E, F), and 168 h (G, H). Yellow arrowheads: spindle-shaped plasmatocytes. Black arrowheads: a collapsed oenocytoid and a spherulocyte, which had probably contaminated the HPO. Red arrowheads: oenocytoids appearing during culture. Blue arrowheads: shrunken cells (inset of panel F is a magnified image of a shrunken cell). All figures are at the same magnification (bar  = 50 µm).

Figure 3. In vitro differentiation into granulocytes and oenocytoids.

A–F: Immunostaining with anti-granulocyte antibody mAb#13 of dispersed HPO cells (A, D), circulating hemocytes from L5D1 larvae (B, E) and HPO cells cultured in plasma-free medium for 7 days (C, F). The photograph shown in (D) was intentionally overexposed in order to detect very weak signals. G–I: Collapse of cultured cells similar to oenocytoids. HPO cells cultured in plasma-containing medium for 7 days were transferred to a glass slide and viewed at 0 min (G), 5 min (H), and 60 min (I). Two of three cells collapsed within 5 min, and the third was unchanged until 50 min later, but finally collapsed at 60 min. J: the cultured cells were incubated in 1% DOPA saline for 2 h. Large cells (arrow heads) turned brown in color. K: cells having properties of both plasmatocytes and oenocytoids were found in the culture of HPO cells.

Figure 4. In vitro differentiation from plasmatocyte to oenocytoid.

When B. mori HPO was cultured in plasma-containing medium, a large number of hemocytes were discharged (A). Discharged hemocytes were spherical or spindle-shaped (B), and approximately 70% of them underwent marked morphological changes after PP treatment (C, D). Non-adherent cells, including immature plasmatocytes and other subsets (D), were washed away. The spread cells, i.e. functional plasmatocytes, were collected and cultured in medium containing larval plasma. Seven days later, oenocytoid-like cells can be seen (E, F). Likewise, functional plasmatocytes from CecB-GFP line were collected, and cultured with discharged cells from non-transgenic larvae. After 7 days, GFP expressing oenocytoid-like cells can be seen (G, H). Bar  = 0.1 mm (A), 40 µm (B, C), 10 µm (E, F), 20 µm (G, H).

Figure 5. Schematic of a hypothesis for hemocyte differentiation in B. mori larva.

1,2: B. mori HPO discharges hemocytes, of which (1) approximately 70% are functional plasmatocytes with (2) the remaining cells putative prohemocytes . 3,4: Prohemocytes discharged into hemolymph could give rise to (3) plasmatocytes and (4) granulocytes . 5: When B. mori larvae are injured, pro-PP is processed into the active form and induces spreading behavior of plasmatocytes . 6: Without inflammation caused by the PP, plasmatocytes can differentiate into oenocytoids (Fig. 4). 7: Oenocytoids are also activated by PP, and release their cellular contents (Supplementary data Fig. S1) including enzymes involved in melanization. 8: Granulocytes differentiated from prohemocytes multiply by mitosis , . 9: Some of the granulocytes may differentiate into spherulocytes . HPO: hematopoietic organ. PR: prohemocyte. PL: plasmatocyte. OE: oenocytoid. GR: granulocyte. SP: spherulocyte.