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.

Immune tolerance to an intestine-adapted bacteria, Chryseobacterium sp., injected into the hemocoel of Protaetia brevitarsis seulensis

“…In addition, peptidoglycan recognition proteins (PGRPs) activate phenoloxidase (PO) in insect hemocytes and activated PO oxidizes phenolic molecules to produce melanin around invading pathogens and wounds 11 . As previously described 12 , phenoloxidase activity in the granulocytes of P. brevitarsis seulensis larvae was detected and also shown to be an important component of the cellular immune reaction by its ability to induce insect hemolymph melanization in various insects 11 13 .…”

“…Activated granulocytes contain phagosomes, lysosomes, and autophagosomes, all of which clear pathogens and engulf potentially hazardous substances 4 . These immune-related morphological changes are also observed in phagocytes from other insects such as flies, mosquitoes, and beetles, although the type of professional phagocyte differs according to the insect species 3 5 12 . Macrophages or phagocytes in vertebrates and invertebrates are the front line of immune defense against virulent pathogens, and recognition of invading pathogens by these cells is the key step in generating immune responses 46 47 .…”

“…Macrophages or phagocytes in vertebrates and invertebrates are the front line of immune defense against virulent pathogens, and recognition of invading pathogens by these cells is the key step in generating immune responses 46 47 . Macrophages or phagocytes engulf potentially hazardous substances, a process that depends on the morphological shape of the pathogen or on expression of certain molecules on the cell membrane 4 12 . To rule out the possibility of a weak cellular immune response induced by Chryseobacterium sp.…”

“…The white-spotted flower chafers, Protaetia brevitarsis seulensis (Coleoptera: Cetoniidae) were reared and maintained as previously described 64 . In addition, the handling procedures of larvae including sample preparation, infection, and image analysis were also performed as previously described 4 12 .…”

Characterization of Qualitative and Quantitative Haemogram Parameters in Insects: Current Concepts and Future Prospects.

“…They distinguished six hemocyte types in these species: PRs, PLs, GRs, SPs, OEs and ADs. Also, Hwang et al (2015) identified six circulating hemocytes in larvae of the Japanese rhinoceros beetle Allomyrina dichotoma: GRs, PLs, OEs, SPs, PRs and ADs. Recently, Chavan et al (2017) identified seven types of haemocytes in haemolymph of the tenebrionid beetle Platynotus belli, viz., PRs, PLs, GRs, OEs, ADs, SPs and CGs.…”