Figure 1.
Photo of (A) Euclea delphinii and (B) Lithacodes fasciola ‘slug’ caterpillars (Limacodidae, Lepidoptera). Photo credit: John Lill.
Figure 2.
Mean hemocyte density in naïve and immune-challenged slug caterpillars.
Hemocyte density (number of hemocytes per ml of hemolymph) from naïve and E. coli-challenged small (white bars) and large (grey bars) size caterpillars of (A) Euclea delphinii and (B) Lithacodes fasciola. Values shown as mean +1 SE, n = 10–28. Different letters above bars in (B) denote significantly different LS means according to Tukey’s HSD test.
Figure 3.
Hemocyte density increases with caterpillar size following bacterial challenge in Lithacodes.
Regression analysis of caterpillar body mass (mg) and hemocyte density (number of hemocytes per ml of hemolymph) in (A) naïve Euclea delphinii, (B) E. coli-challenged Euclea delphinii, (C) naïve Lithacodes fasciola, and (D) E. coli-challenged Lithacodes fasciola caterpillars. Each data point represents an individual caterpillar.
Figure 4.
Monolayers prepared from hemocytes extracted from slug caterpillars.
Panels (A) and (C), differential interference contrast and fluorescent images of hemocytes extracted from Euclea delphinii. Panels (B) and (D), hemocytes extracted from Lithacodes fasciola. Hemocytes were fixed and stained with phalloidin (green) cytoskeleton stain and Hoestch 33342 (blue) nuclear stain to reveal cell morphology. Granulocytes (Gr) and plasmatocytes (Pl) displayed spreading and adhesive behavior whereas oenocytoids (Oe) appear to be non-adhesive. Scale bar = 25 µm.
Figure 5.
Hemocytes from Euclea and Lithacodes belong to three distinct morphotypes.
(A). Hemocyte types from Euclea delphinii: (a) oenocytoids (Oe), (c) granulocytes (Gr), and (e) plasmatocytes (Pl). For comparison, hemocytes extracted from Lithacodes fasciola are shown alongside: (b) oenocytoids (Oe), (d) granulocytes (Gr), and (f) plasmatocytes (Pl). (B) Percentages of the different hemocyte types collected from Euclea and Lithacodes caterpillars, shown as mean ± SE. Cell counts were calculated from six field views per preparation (n = 4 animals per species). A total of 1490 hemocytes from Euclea and 961 from Lithacodes were counted. Granulocytes were the most abundant followed by plasmatocytes and oenocytoids. Scale bar = 10 µm.
Figure 6.
Granulocytes and plasmatocytes from Euclea and Lithacodes display phagocytic activity.
(A) Hemocyte cells from Euclea delphinii and Lithacodes fasciola are capable of phagocytosing beads (a, b) and E. coli bacteria (c, d). Image overlays show hemocytes containing phagocytosed beads (green) in (a) Euclea and (b) Lithacodes as well as labeled E. coli (red) inside phagocytic cells in (c) Euclea, and (d) Lithacodes. Inset in (d) shows non-fluorescent extracellular bacteria. (B) Percentage of phagocytosis-positive cells for each hemocyte type, shown as mean ± SE. Phagocytosis-positive cells were counted from six different field views per animal (n = 4) for both species. Granulocytes (Gr) were significantly more efficient at engulfing bacteria compared to plasmatocytes (Pl) in both Euclea (t = 3.805, df = 6, P = 0.0089) and Lithacodes (t = 3.093, df = 6, P = 0.0213), whereas oenocytoids (Oe) are not phagocytic. ** = P<0.01; * = P<0.05; Scale bar = 25 µm.