Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.

Our results indicate a conserved role for GPHR family receptors in controlling germline development and fertility. Our data suggest a model whereby FSHR-1 signaling acts in parallel to the known sex-determination pathway to control multiple aspects of germline development.

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.

To isolate antimicrobial-related genes from the beet armyworm, Spodoptera exigua, we performed GeneFishing, a polymerase chain reaction (PCR)-based differential display technique. An attacin-like complementary DNA (cDNA) including a 3'-untranslated region was identified from among 18 over-expressed genes in microbial-infected larvae. The full-length attacin cDNA from S. exigua cDNA (Seattacin) was cloned using rapid amplification of cDNA ends PCR. The attacin-like cDNA transcript was 765 nucleotides in length, and the predicted polypeptide was 254 amino acids in length with a calculated molecular mass of 27.6 kDa and an isoelectric point of 6.44. The protein sequence of the attacin-like cDNA showed high identity to that of Trichoplusia ni (61.2%). The amino acid sequence identity of Seattacin to the orthologous proteins in Bombyx mori, Manduca sexta, Heliothis virescens, Hlicoverpa armigera, Hyphantria cunea, Hyalophora cecropia, and Drosophila melanogaster was 61.2, 46.1, 44.5, 42.2, 39.5, 45.1, and 24.0%, respectively. To examine possible immune functions of the attacin-like cDNA, its expression was investigated by reverse transcriptase PCR analysis after challenging S. exigua with microorganisms. The attacin-like cDNA was expressed at high levels 12 h post-infection, and its expression was slightly induced 4-8 h post-infection compared to control larvae inoculated with sterile water. Furthermore, induced Seattacin showed biological activity against several bacteria including Escherichia coli DH5α, Pseudomonas cichorii, Bacillus subtilis, and Listeria monocytogenes. These results suggest that the attacin-like cDNA of S. exigua codes for antimicrobial peptides.

In this study, more than 1,000 cricket (Gryllus bimaculatus) hemocytes were classified based on their size and morphology. These hemocytes were classified into six types: granulocytes, plasmatocytes, prohemocytes, spherulocytes, coagulocytes, and oenocytoids. Hemocyte cultures was observed in real time to determine which hemocytes were associated with cellular immune responses against potential pathogens. Granulocytes were identified as the professional immune cell that mediates nodulation, encapsulation, and phagocytosis of pathogens. Granulocytes have been shown to actively produce various sticky nets (amoeba-like hairs and extracellular traps) from their plasma membranes that they use to gather other hemocytes and to implement cellular immune responses. The activation of lysosomes in granulocytes started at 4 h, peaked at 12 h, and returned to baseline by 24 h post-infection. At 48 h post-infection, cells could be found within the cytoplasm of granulocytes and reactivated lysosomes surrounding these cells were visible. This result seems to reflect a phenomenon in which necrotic granulocytes are removed by other healthy granulocytes. This unique mechanism of cellular immunity is therefore a way to efficiently and effectively remove pathogens and simultaneously maintain healthy hemocytes.