Insect cellular immune reaction, which generally includes phagocytosis, encapsulation and nodule formation, is achieved by hemocytes circulating in insect haemolymph. The shift of hemocytes from the normal phase to the adhering phase is an important process in the cellular immune reaction, which includes the attachment of hemocytes to foreign surfaces or other hemocytes via adhesion factors. Neuroglian is one of the adhering factors associated with encapsulation in Manduca sexta and Drosophila melanogaster. Here we studied the localization of neuroglian (MsNrg) in Mythimna separata and its functional role in the cellular immune reaction. The distribution of MsNrg mRNA between hemocyte populations was determined using real-time quantitative reverse transcription PCR and in situ hybridization, which revealed that MsNrg was highly expressed in adhering hemocytes, especially in plasmatocytes. Unexpectedly, the transcript was observed as well in non-adhering hemocytes, implying neuroglian has a function in non-adhering hemocytes. Moreover, we showed that the amount of MsNrg mRNA was not changed by injections of either biotic or abiotic non-selves. Fewer latex beads were fully encapsulated by hemocytes in larvae treated with MsNrg double-stranded RNA than in control larvae, but their ability to achieve phagocytosis and nodule formation remained unchanged by the MsNrg knockdown. These results indicate that the function of neuroglian in the cellular immune reaction is conserved in D. melanogaster and lepidopteran species, and neuroglian in non-adhering hemocytes could possess unidentified function., Kakeru Yokoi, Yoshiaki Kato, Masahiro Suzuki, Ken Miura., and Obsahuje bibliografii
Coordinated regulation of apoptosis is critical for development, homeostasis, and immunity in larvae of Metazoa. We determined the full nucleotide sequence of an inhibitor of an apoptosis protein in a lepidopteran insect Mythimna separata (Walker) (MsIAP) and carried out functional analyses of the MsIAP. The full-length cDNA of MsIAP was 1642 bp, which encoded 379 amino acid residues with a calculated molecular mass of 41,834 Da, and two BIR domains and one RING domain revealed using amino acid sequence analysis. In addition, the sequences of these domains were similar to Drosophila IAP1 and those of some other lepidopteran insects. We carried out a functional analyses of MsIAP related to apoptosis regulation using RNA interference. The effects of MsIAP knockdown on adhering hemocytes and non-adhering hemocytes as controls were examined using Hoechst33342/propidium iodide staining, effector caspase activity and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. A significantly higher number of propidium iodide and TUNEL-positive cells was recorded in adhering hemocytes from MsIAP knockdown larvae than from control larvae, but these differences were not recorded for non-adhering hemocytes. However, higher effector caspase activity was detected in both adhering and non-adhering hemocytes from MsIAP knockdown larvae compared to that in control larvae. These results indicate that the knockdown of MsIAP induces apoptosis in larval adhering hemocytes, which MsIAP negatively and non-redundantly regulate apoptosis, and that IAP function is conserved in M. separata and other insect species including Drosophila and several lepidopteran insects., Masashi Kamezaki, Kakeru Yokoi, Ken Miura., and Obsahuje bibliografii