Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) and the Cellular Immune Response to Microorganisms

Abstract

Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36%) compared to controls (5.08%), as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3) to the phagosome, which may result in enhanced pathogen killing.

Fig 1. Image of hemocytes.

Hemocytes were classified as prohemocytes (A, A-1, and A-2), oenocytoids (B, B-1, and B-2), adipohemocytes (C, C-1, and C-2), spherulocytes (D, D-1, and D-2), plasmatocytes (E, E-1, and E-2), and granulocytes (F, F-1, and F-2) on the basis of their size and morphology. Confocal images of hemocytes stained with DAPI (blue) to label nuclei and antibodies to filamentous actin (F-actin; red) to label the cytoskeleton. N, nucleus. (A-F1) Scale bar = 20 μm, (A2-F2) Scale bar = 2 μm.

Fig 2. Image of immunologically activated granulocytes, including phagocytosis and nodulation.

(A, B, and C) The granulocytes changed their shape and generated lodopodia-like or fan-like structures (indicated by white arrow). The vacuoles expanded in the cytoplasm (indicated by red arrows). Several filopodia were also generated in plasmatocytes (D; indicated by yellow arrows). The granulocytes phagocytosed and engulfed GFP-expressing yeast at 30 min post-infection (E and F). Nodulation by granulocytes at 30 min post-bacterial infection (G and H). N, nucleus; GR, granulocyte; AD, adipohemocyte. Scale bar = 20 μm.

Fig 3. Total hemocyte and average proportions of hemocytes in native and challenged larvae.

Total hemocyte counts (A and B) and differential hemocyte counts (A-1 and B-1) were performed. The number of hemocytes of the six circulating hemocyte types were counted in 39 larvae (33,270 hemocytes). Each group (control larvae or 2, 4, 6, 8, 12, or 24 h post-infection) contained three larvae. Results are given as the mean and standard deviation.*(P<0.05) Challenged larvae were infected with bacteria (panel A and A-1) or yeast (panel B and B-1). PR, prohemocytes; PL, plasmatocytes; GR, granulocytes; AD, adipohemocytes; SP, spherulocytes; OE, oenocytoids.

Fig 4. LysoTracker Red labeling of lysosomes in granulocytes and flow cytometric analysis after bacterial infection.

(A and A-1) 0 h post injection, (B and B-1) 4 h post injection, (C and C-1) 12 h post injection. A1, B1, and C1 indicate a higher magnification of the regions in inset of panel A, B, and C. Greater than 90% of the granulocytes were stained with LysoTracker at 4 h post-bacterial infection, while very little LysoTracker staining was observed in control larvae. The red signal returned to baseline at 12 h post-infection. (D through H) flow cytometric analysis at 0 h, 2 h, 4 h, 6 h, and 12 h post injection. Based on the red fluorescence intensity, two peaks were identified, negative and positive peak. (I) The black line histogram indicates control larva hemocytes and the red line histogram indicates bacterial-challenged larva hemocytes at 4 h post injection. N, nucleus; GR, granulocytes; AD, adipohemocytes. Scale bar = 20 μm.

Fig 5. GFP-LC3 labeling in granulocytes and flow cytometric analysis after bacterial infection.

A Cyto-ID Autophagy Green dye reagent was used for the detection of autophagosome formation in granulocytes at 0, 4, 8, 12, and 24 h post infection. (A, B and C) Confocal Images of granulocytes stained with DAPI and GFP-LC3 (A; 0 h post injection, B; 8 h post injection, C; 24 h post injection); and flow cytometric analysis at 0, 4, 8, 12, and 24 h (D through H). After 8 h of infection, positive staining was observed in the granulocytes (B) and the GFP signal was weakly maintained at 24 h post infection (C). Based on the green fluorescence intensity, two peaks were identified, negative and positive peak. The increase in GFP-LC3 staining at 8 h post infection (F). (I) Representative histogram overlay (black line; 0 h post infection, green line; 8 h post infection). N, nucleus; GR, granulocytes; AD, adipohemocytes. Scale bar = 20 μm.