Characterization of the immune hemocyte in larvae of Dorcus titanus castanicolor (Motschulsky, 1861) (Lucanidae, Coleoptera)

There are four types of hemocytes in the stag beetle hemolymph: granulocytes, prohemocytes, coagulocytes, and spherulocytes. Unusually, the size and shape of the hemocyte subtypes are similar. To identify the predominant immune hemocytes, we injected carboxylate‐modified polystyrene latex beads and E. coli particles into the hemocoel of stag beetles. In response to the pathogens, only granulocytes underwent morphological changes, including the extension of fan (or amoeba)‐like structures from their membrane, and had bacterial particles in their cytoplasm. At 6 h post‐injection, lysosomes in the cytoplasm of the granulocytes were activated to remove the pathogens. At 12 h post‐injection, more lysosomes were activated along with phagocytosis of the E. coli particles. At 24 h to 48 h post‐injection, the bacterial particles, including activated lysosomes, were gradually cleared from the cytoplasm. To confirm these results, we performed flow cytometry analysis. Consistent with our microscope observations, the granulocytes were activated after 6 h post‐infection, and lysosomes were mostly activated at 12 h. These results suggest that bacteria are actively engulfed between 6 and 12 h post‐infection by granulocytes. Granulocyte lysosomes were activated within 12 h to digest the bacteria, which were completely cleared by 48 h post‐infection.